Apparatus for capillary electrophoresis comprising polyelectrolyte multilayers

ABSTRACT

An apparatus and method for capillary zone electrophoresis includes a polyelectrolyte multilayer positioned in a capillary tube for analytical separations of macromolecules. The capillary comprises a passage defined by passage walls comprising fused silica. The polyelectrolyte multilayer is positioned within the passage adjacent the walls, and comprises an organic polyelectrolyte. The passage may further comprise nonporous silica particles coated with a multilayer including a plurality of polyelectrolyte layers. An apparatus includes a power supply having a positive electrode and a negative electrode for generating an electric field therebetween. The apparatus includes a capillary having a passage formed by passage walls and comprising therein a polyelectrolyte multilayer positioned substantially within the passage. The passage has a first end electrically connected to the positive electrode and a second end electrically connected to the negative electrode to thereby generate an electric field through the passage. The apparatus also includes a sensor positioned adjacent the passage for sensing macromolecules.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/933,179, filed on Sep. 2, 2004, now U.S. Pat. No. 7,285,421, which isa continuation of U.S. application Ser. No. 10/145,161, filed on May 14,2002, now U.S. Pat. No. 6,841,054, which is a divisional U.S.application Ser. No. 09/442,198, filed on Nov. 16, 1999, now U.S. Pat.No. 6,402,918, which claims benefit of Provisional Application No.60/108,528, which was filed on Nov. 16, 1998, a date to which priorityis claimed under 35 U.S.C. § 120 and 37 CFR § 1.78, and whichProvisional Application is incorporated by reference herein in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

The claimed invention was made with financial support from the UnitedStates Government and the inventors hereby acknowledge that thegovernment may have certain rights in the invention, as specified bylaw.

FIELD OF THE INVENTION

The present invention relates to the field of molecular separations and,more particularly, to molecular separation by electrophoresis with apolyelectrolyte multilayer positioned within a very small passage suchas in a capillary tube.

BACKGROUND OF THE INVENTION

Electrophoresis is a method for separation of individual molecularspecies from a mixture by the application of an electric field. Thetechnique relies on the migration of charged molecules through asolution in the electric field. Separation of the molecules occurs dueto their different rates of movement through the solution, the ratebeing influenced by factors such as the pH of the solution, the mass andcharge of the molecule, and the strength and duration of the electricfield.

The electrophoretic separation may be carried out in a support mediumwherein the molecules to be separated are loaded. Common support mediafor electrophoretic molecular separation include gels of variouschemical formulations and physical configurations. Support gels,however, may be difficult to prepare, handle, and process, therebyresulting in reproducibility problems.

One approach for increasing reproducibility has been the use ofcapillary tubes, but without a support medium for the separation, otherthan the electrophoresis buffer itself. A capillary tube for use in thistechnique is substantially a small tube having a void space in the formof a very narrow passage therein. The electrophoretic separation iscarried out within the narrow passage. For example, in the late fiftiesHjerten reported success in electrophoretic molecular separations usinga quartz capillary tube having an internal diameter of about 1-3 mm andusing only a suspending solution as the separation medium. Hjerten, S.,Arkivkem, 1958, 13, 151. Hjerten's system was never commercialized dueto problems related to complex design and insufficient heat dissipationduring the process. Over the succeeding years other authors reportedimproved separations and increased heat dissipation using thinnercapillaries. In addition, what may be considered the first apparatus forcapillary zone electrophoresis was described by Jorgenson and Lukacs.See Jorgenson, J, and K. D. Lukacs, Anal. Chem., 1981, 53, 1298; andScience, 1983, 222, 266.

As known in the prior art, capillary zone electrophoresis (CZE) isgenerally performed as follows. An apparatus for CZE preferably includesa power supply which may provide for reversing polarity, the powersupply being connected by each of two electrodes to each of two bufferreservoirs. A fused silica capillary is positioned so as to form aconnecting bridge between the two reservoirs. The capillary is generallyfrom about 20 cm to 1 m long, and includes a passage of from about 25 to100 μm internal diameter. The capillary generally has an outer layer ofpolyimide to provide added flexibility, as well as durability. Detectionof molecular species is performed in an area, or window, of thecapillary where the polyimide coating has been stripped away. Suitabledetection methods include absorbance, laser-induced fluorescence,refractive index conductivity, electrochemical detection, and even massspectrometry, although this last approach requires an interface otherthan the capillary tube.

A sample containing the molecular species to be separated may generallybe introduced in the capillary either hydrodynamically orelectrokinetically. Those skilled in the art will know that hydrodynamicinjection of the sample may be variously accomplished. The capillary maybe elevated at one end to inject the sample by substantially syphoningit into the passage. A sample vial may be positioned in fluid connectionwith the passage, and fluid pressure may be applied to the capillary orto the sample vial to thereby move the sample into the passage.Conversely, suction may be applied at a second end of the passage todraw sample from a sample vial connected to a first end of thecapillary. Injection may also be accomplished by means of a syringe, andmay preferably include a sample splitter. Electrokinetic injectionrelies on the application of an initial voltage through the passage toinitiate sufficient fluid flow to bring the sample into the passage,thereafter initiating predetermined electrophoretic separationconditions.

Commercially available systems for CZE also include features forrinsing, and for added heat dissipation. Rinsing is accomplished byflushing a rinse fluid through the passage, the rinse fluid usuallybeing water, a buffer, or another predetermined solution. Rinse cyclesmay be effected by applying pressure to the system to thereby flush therinse fluid through the microchannel. For added heat dissipation,commercial systems include a coolant feature. For example a fluorocarbonfluid may be used to bathe the capillary so as to prevent uneven heatdissipation during the electrophoresis.

Molecular separation by electrophoresis relies on the electricalinteractions affecting the molecular species being separated. Thepassage walls defining the passage have naturally occurring electricalcharges on their surfaces. In a fused silica capillary, for example,surface silanol groups (Si—OH) are substantially deprotonated at a pHabove 2, the wall thereby having negative charges on its surface. Atightly adsorbed, substantially stagnant layer of cations from a fluidcontained in the passage will localize adjacent the negatively chargedwall so as to partially neutralize the negative charge on the wall. Theremaining negative charge on the wall is neutralized by excess cations,which remain in the fluid in a more diffuse layer of mobile, solvatedcations. The electrical potential across the double layer comprising thewall and the cations is known in the art as “zeta potential”. In anelectric field, cations are attracted to the cathode, and anions areattracted to the anode. In CZE, the cations in the diffuse layer migratetoward the cathode and, since they are solvated, pull solvent moleculesalong in their migration, creating a flow of solvent. This solvent flowinduced by the electric field, is known as electroosmotic flow (EOF).The velocity of the EOF may be calculated according to equations wellknown in the art. During electrophoresis, molecules are separated by theEOF in relation to their charge and size. Because fluid flow isgenerally toward the cathode, molecules tend to elute (be released) fromthe capillary cations first, followed by neutral molecules havingsubstantially no net charge, followed by anions. Neutral molecules tendnot be separated from each other. Various factors may affect thevelocity of the EOF, and hence also affect molecular separation. Factorsaffecting EOF velocity and molecular separation include viscosity of thesuspending fluid, particularly adjacent the passage wall, a change inthe electrical charge of the wall itself, or alterations to theneutralizing charges overlying the wall.

Polyelectrolytes have been previously used for modifying theelectrophoretic properties of fused silica capillary passages.Adsorption of a cationic polyelectrolyte to the negatively chargedsilica surface effectively reverses the surface charge from negative topositive. This charge reversal causes fluid flow to be toward the anodeso that anions elute first, followed by neutral molecules, followedlastly by cations. Polyelectrolytes previously used to coat silicasurfaces include polyarginine, chitosan, poly (diallyldimethylammoniumchloride) (PDADMAC), and polyethylenimine. Prior electrophoretictechniques have employed single layers of polyelectrolyte.

A method for forming multilayers of polyelectrolytes has now beendescribed. Decher, G. and J. Schmitt, J. Prog. Colloid Polym. Sci.,1992, 89, 160; and Decher, G., Science, 1997, 277, 1232. However, theadvantages of polyelectrolyte multilayers for capillary electrophoresishave not been recognized before the present invention.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a capillary tube having a multilayer comprising apolyelectrolyte and positioned for analytical separations of molecules.

It is an object of the invention to provide increased electrophoreticefficiency, and substantially equal efficiency at pH of about 4 andabout 6.

It is a further object of the invention to provide substantiallyreproducible electroosmotic mobility among capillaries manufacturedusing the same procedure.

It is yet another object of the invention to provide a capillary coatedwith a polyelectrolyte multilayer which may be used for many analyticalcycles while yielding substantially reproducible results.

It is a further object of the invention to provide a coated capillarywhich substantially reduces irreversible adsorption of large polyionssuch as proteins to the passage wall.

It is an additional object of the invention to provide a coatedcapillary which is easily manufactured.

It is also an object of the invention to provide a coating forelectrophoretic separations which also functions as a partition mediumallowing separation of neutral and/or hydrophobic analytes.

It is still another object of the invention to provide a capillary zoneelectrophoresis system which requires no pre-analysis equilibration, sothat a relatively stable electroosmotic flow is obtained substantiallymore rapidly.

Accordingly, the capillary tube comprises a generally cylindrical voidspace, or passage, having a lengthwise dimension and a cross sectiondimension of from about five micrometers to about one hundredmicrometers. The multilayer comprising a plurality of polyelectrolytelayers is positioned within the cylindrical void adjacent the walls. Thecapillary tube may preferably comprise a plurality of layers of acationic polyelectrolyte and an anionic polyelectrolyte.

An embodiment of the invention includes a plate having a multilayer foranalytical separation of macromolecules. The plate comprises a passagesubstantially defined by passage walls, and a multilayer positionedwithin the passage adjacent the walls, the multilayer comprising aplurality of polyelectrolyte layers. The passage may preferably bepositioned within a capillary tube or within a plate. In addition, theplate may comprise a plurality of passages. The passage preferablycomprises walls of fused silica.

In yet another embodiment of the invention, the passage coated with thepolyelectrolyte multilayer may further comprise particles coated withpolyelectrolyte multilayers. The particles may preferably comprisenon-porous silica in approximate sizes from about 1-5 μm, but may alsocomprise other suitable materials. Presence of these multilayer coatedparticles improves separation of neutral molecules by increasingtransport of molecular species from the fluid flow into the multilayer.Multilayer coated particles may be included in any of the otherembodiments of the present invention, for example in a capillary, or aplate. In addition, the coated particles may also be included in anapparatus embodiment of the invention.

The present invention also includes an apparatus for electrophoreticseparation of macromolecules. The apparatus comprises a power supplyhaving a positive electrode and a negative electrode for generating anelectric field; a multilayer positioned substantially in a passageformed by passage walls, the passage having a first end electricallyconnected to the positive electrode and a second end electricallyconnected to the negative electrode to thereby generate an electricfield through the passage, and wherein the multilayer comprises aplurality of polyelectrolyte layers; and a sensor positioned adjacentthe passage for sensing macromolecules.

The invention further includes a method for analytical separation ofmacromolecules. The method comprises the step of forming a passagedefined by passage walls. A second step in the method includespositioning a multilayer substantially within the passage adjoining thewalls, wherein the multilayer comprises a plurality of polyelectrolytelayers. A third step includes positioning a sample containingmacromolecules substantially within the passage. A fourth step includesgenerating a flow of a predetermined fluid through the passage tothereby substantially separate macromolecules from the sample responsiveto an interaction with the multilayer. The flow of fluid may preferablybe generated by passing an electric field through the passage, alsoknown as electrophoresis, or by applying pressure to thereby generatethe fluid flow.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings inwhich:

FIG. 1 is a schematic diagram of a capillary tube having a passage wallcoated with a polyelectrolyte multilayer according to an embodiment ofthe present invention;

FIG. 2 is a schematic diagram of a microchannel plate;

FIG. 3 is a schematic view of the capillary tube passage of FIG. 1including multilayer coated particles;

FIG. 4 is a schematic diagram of the apparatus of the present invention;

FIG. 5 shows electrophoretic separation of several proteins using asingle layer of polyelectrolyte (PDADMAC) at about pH 4, as described inExample 1;

FIG. 6 shows electrophoretic separation of several proteins using amultilayer of polyelectrolytes at about pH 4, as described in Example 1;

FIG. 7 shows electrophoretic separation of several proteins using asingle layer of polyelectrolyte (PDADMAC) at about pH 6, as described inExample 2;

FIG. 8 shows electrophoretic separation of several proteins using amultilayer of polyelectrolytes at about pH 6, as described in Example 2;

FIG. 9 shows electro-chromatographic separation of two neutral moleculesand comparing a single layer of polyelectrolyte with a multilayer, asdescribed in Example 3;

FIG. 10 shows electro-chromatographic separation of four neutralmolecules using a multilayer, as described in Example 3;

FIG. 11 shows separation of acetone and naphthalene using a multilayer,as described in Example 3;

FIG. 12 shows the separation described in Example 4; and

FIG. 13 is a flow chart illustrating the method of the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theillustrated embodiments set forth herein. Rather, these illustratedembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art. Like numbers refer to like elements throughout, andprime notation when used indicates similar elements in alternativeembodiments.

As illustrated in FIG. 1, the invention discloses an apparatus andmethod for capillary zone electrophoresis. The invention includes apolyelectrolyte multilayer 21 positioned adjacent the walls 23 of thepassage 25, preferably positioned in a capillary tube or a microchannelplate. Those skilled in the art will know that a microchannel plateincludes one or more very small passages extending through and enclosedwithin the plate so as to be a substantial equivalent of the passage ofa capillary tube. The multilayer 21 positioned within the passage 25provides various advantages in analytical molecular separations, andparticularly in analytical molecular separations by electrophoresis.

A preferred embodiment of the invention includes a capillary tube 27having a passage 25 defined by passage walls 23 comprising fused silica.The capillary 27 includes a multilayer 21 positioned within the passage25 adjacent the walls 23, the multilayer comprising a plurality ofpolyelectrolytes forming layers, as shown schematically in FIG. 1. Thoseskilled in the art will readily recognize that a polyelectrolyte isgenerally a polymer, and particularly an organic polymer, having apermanent or pH-dependent charge. In addition, the skilled artisan willunderstand that a polyelectrolyte layer pair includes two alternatingpolyelectrolytes of complementary charge, positive and negative as shownin FIG. 1, coupled by the interaction of those charges, the coupled pairof polyelectrolytes forming one layer pair of the multilayer.

Preferred polyelectrolytes in the present invention includepoly(diallyldimethylammonium chloride) (PDADMAC), and poly(styrenesulfonate), sodium salt (PSS). The capillary tube 27 having themultilayer 21 is preferably employed for separating macromolecules byelectrophoresis. Those skilled in the art will additionally understandthat the term “macromolecule” as used herein is intended to generallyinclude any complex molecule, and particularly those having biologicalorigin and/or biological importance, such as nucleic acids,polypeptides, proteins, enzymes, antigenic molecules, antibodies,polymers, drug molecules, and other natural and synthetic molecules.

In the capillary tube 27, the passage 25 preferably comprises asubstantially cylindrical void space having a diameter of from aboutfive micrometers to about one hundred micrometers. The capillary tube 27includes a first end, a second end, and a lengthwise dimension extendingtherebetween, wherein the passage 25 extends along the lengthwisedimension from a first opening positioned at the first end to a secondopening positioned at the second end.

The multilayer 21 positioned within the capillary 27 preferably furthercomprises a plurality of layers of an organic cationic polyelectrolyte.In another embodiment the organic polyelectrolyte preferably comprises aplurality of layers of an organic anionic polyelectrolyte. Themultilayer 21 may additionally comprise a plurality of organicpolyelectrolytes. Additionally, the multilayer 21 in the capillary tube27 also preferably comprises alternating layers of an organic cationicpolyelectrolyte and an organic anionic polyelectrolyte, as shownschematically in FIG. 1. Furthermore, concentration of thepolyelectrolyte may vary within the multilayer. In other preferredembodiments of the capillary tube 27, the multilayer 21 may be depositedin the presence of sodium chloride, and may comprise sodium chloride.The multilayer 21 may also preferably comprise a modifier such as anorganic solvent, for example ethanol, and may comprise a weakpolyelectrolyte. Capillary tubes comprising a polyelectrolyte multilayerin the present invention are prepared as described in the scientificpaper published by Graul, T. W. and J. B. Schlenoff, in AnalyticalChemistry, 1999, 71, 4007-4013, which is hereby incorporated byreference in its entirety.

Yet another preferred embodiment of the invention, as shown in FIG. 2,includes a microchannel plate 31 for analytical separation ofmacromolecules. The plate 31 comprises a passage 25′ substantiallydefined by passage walls. Those skilled in the art will appreciate that,although the passage is shown generally cylindrical in theseillustrations, the passage may be formed in many other shapes. Inaddition, the artisan will know that the plate may be fabricated byvarious processes, including forming two halves, each half having halfof a passage cut into it, the half plates being joined so as to form acomplete plate enclosing the passages therein. The microchannel platepassage 25′ includes a multilayer 21′ positioned within the passageadjacent the walls 23′, wherein the multilayer comprises an organicpolyelectrolyte. The passage 25′ within the plate 31 may comprise a voidspace having a predetermined lengthwise dimension and a cross sectiondimension of from about five micrometers to about one hundredmicrometers. The passage in the plate may comprise a generallycylindrical void space, such as that of a capillary tube. The passage inthe plate also preferably comprises a first end, a second end, thelengthwise dimension extending therebetween, and the passage extendingalong the lengthwise dimension from a first opening positioned at thefirst end to a second opening positioned at the second end. In anotherembodiment, the plate preferably comprises fused silica. Additionally,and as shown in FIGS. 2 a and 2 b, the plate 31 may further comprise aplurality of passages 25′, a particularly useful configuration forrunning multiple separations simultaneously. However, those skilled inthe art will recognize that the passages of the present invention may beformed from any of a variety of materials known to be suitablesubstrates for such analytical separations, and particularly forelectrophoresis.

The multilayer 21, whether positioned within the passage of a capillaryor plate, may be formed so that it includes various other components.For example, the multilayer may be deposited in the presence of sodiumchloride, and may comprise sodium chloride. The multilayer may alsoinclude a modifier, particularly an organic solvent such as ethanol.Inclusion of such components preferably produces a thicker multilayer,or may be used to advantageously provide other useful properties to themultilayer. In addition, the character of the polyelectrolyte within themultilayer 21 may be selected to produce multilayers having differentpredetermined capabilities. For example, the multilayer may comprise oneor more weakly dissociating polyelectrolytes, so as to control thecharge of the polyelectrolyte, and thereby the charge of the multilayer,by the pH of the solution.

In a further embodiment of the invention, shown in FIG. 3, the passage25 comprising the polyelectrolyte multilayer 21 may be filled withparticles 35 ranging in size from about 1-5 μm, the particles themselvescoated with polyelectrolyte multilayers 21″. The particles 35, asillustrated schematically in FIG. 3, preferably comprise non-poroussilica but may also comprise other suitable materials. Presence of thesemultilayer coated particles 35 improves separation of neutral moleculesby increasing transport of molecular species from the fluid flow intothe multilayer. These multilayer coated particles 35 may be included inany of the other embodiments of the present invention, for example in acapillary, or a plate. In addition, the coated particles may also beincluded in an apparatus 41 embodiment of the invention, as describedbelow.

Another aspect of the invention includes an apparatus 41 forelectrophoretic separation of macromolecules. As illustrated in FIG. 4,the apparatus 41 includes a power supply 43, a multilayer positioned inthe passage of a capillary tube or plate, and a sensor 45. FIG. 4illustrates the apparatus 41 embodiment having a capillary 27 comprisingthe multilayer 21 as illustrated in FIG. 1. The multilayer 21 positionedin the passage 25 comprises an organic polyelectrolyte. The power supply43 includes a positive electrode 47 and a negative electrode 49 forgenerating an electric field therebetween. The capillary 27 passage hasa first end electrically connected to the positive electrode 47 and asecond end electrically connected to the negative electrode 49 tothereby generate an electric field through the capillary 27 passage. Thesensor 45 is positioned adjacent the capillary 27 passage for sensingmacromolecules, thereby providing information for monitoring theseparation.

In the apparatus 41 of FIG. 4, the multilayer is positioned within thepassage substantially adjacent the passage walls, and preferablycomprises a plurality of organic polyelectrolytes, as shown in FIG. 1.The passage 25 preferably comprises a void space, which may be shaped asa substantially cylindrical passage having a diameter of from about fivemicrometers to about one hundred micrometers. In a preferred embodimentof the apparatus, as shown in FIG. 4, a capillary tube 27 comprises thepassage 25. Alternatively in the apparatus 41, a plate 31 may comprise asingle passage, and preferably comprises a plurality of passages 25′, asshown in FIG. 2, for running multiple separations simultaneously. As inother embodiments of the invention, in the apparatus 41 the passagewalls 23 in either a capillary or plate preferably comprise fusedsilica. Other aspects of the multilayer included in the apparatus are asdescribed herein for multilayers positioned in a capillary or a plate.

A method aspect of the present invention is illustrated in the flowdiagram shown in FIG. 13. The method from the start (Block 51) includesthe step of forming a passage (Block 53) defined by passage walls. Afurther step includes positioning a multilayer substantially within thepassage (Block 55) and adjoining the walls, wherein the multilayercomprises an organic polyelectrolyte. An additional step includesplacing a sample containing macromolecules substantially within thepassage (Block 57). A further step is generating a flow of apredetermined fluid (Block 59) through the passage to therebysubstantially separate macromolecules from the sample responsive to aninteraction with the multilayer. Preferably, in the generating step thefluid is substantially electrically conductive and the flow is generatedby applying an electric field through the passage, a process known inthe art as electrophoresis. In another preferred embodiment of themethod, the flow is generated by applying fluid pressure through thepassage, the macromolecules carried by the flow being separatedresponsive to an interaction with the multilayer. Those skilled in theart will realize that fluid pressure may be generated by applying acompression force or a vacuum force to the fluid.

An additional preferred embodiment of the method further comprises thestep of sensing the fluid flow (Block 61) to thereby substantially senseseparated macromolecules, whereafter the method stops (Block 63). Thestep of sensing may be performed by any one of several methods known inthe art, for example, absorbance, laser-induced fluorescence, refractiveindex conductivity, electrochemical detection, and mass spectrometry.

EXPERIMENTAL EXAMPLES

Experimental examples are set forth below in some detail to furtherillustrate the various features and advantages of the present invention,and as an aid to those skilled in the art in understanding theinvention.

General Procedures

In general, the materials and methods in the experimental examples areas set forth below and as described by Graul, T. W. and J. B. Schlenoff,in Analytical Chemistry, 1999, 71, 4007-4013.

Poly(diallyldimethylammonium chloride), PDADMAC (Aldrich,M_(W)=250,000-400,000, M_(W)/M_(n)=2.9) and poly(styrene sulfonate),sodium salt, PSS (Scientific Polymer Products, M_(W)=6×10⁶) werepurified by extensive dialysis against distilled water using 12-14,000molecular-weight-cutoff dialysis tubing (Allied-Fisher Scientific).Basic proteins—Chymotrypsinogen A (Type II from Bovine Pancreas),Ribonuclease A (Type XII-A from Bovine Pancreas), Cytochrome C (fromBovine Heart), and Lysozyme (Grade I from Chicken Egg White) werepurchased from Sigma-Aldrich (St. Louis, Mo.) and used as received.Protein sample concentrations before injection were either 0.30 or 0.60mg mL⁻¹. All other chemicals were used as received from Allied-Fisher.

Separations were performed on a Beckman P/ACE System 2100 CapillaryElectrophoresis unit (Palo Alto, Calif.) with UV detection. Fused silicacapillary with 50 μm I.D., 360 μm O.D., and polyimide outer coating waspurchased from Polymicro Technologies (Phoenix, Ariz.).

Multilayer coatings were deposited in the capillary tube using the rinsefunction (rate of 250 cm min⁻¹ linear velocity, 5 μL min⁻¹ volume flowrate) on the Beckman CE system. Polymer deposition solutions contained10 mM polymer, and varying NaCl concentration (polymer concentrationsare based on the repeat unit). The capillary was first conditioned by a30 min rinse of 1 M NaOH. Then water was flushed through the capillaryfor 3 min. The first monolayer of polymer (PDADMAC) was deposited byrinsing the polymer solution through the capillary for 20 min followedby a 5 min water rinse. All other polymer depositions were done with 5min rinses followed by 5 min water rinses.

Multilayer coatings used for the protein separations and reproducibilitystudies comprised six and a half layered pairs (a layered pair generallycomprises a layer of a cationic polyelectrolyte and a layer of anionicpolyelectrolyte, also termed a “bilayer” in other studies), where thefirst 3.5 layer pairs were deposited with no salt present and the lastthree were deposited with 0.5 M NaCl present.

Nonporous silica particles of about 4.5 μm diameter were coated withpolymer by the following method. The particles were suspended in asolution containing 10 mM polymer and 100 mM NaCl. The particles weresettled out of suspension with the aid of a centrifuge. The polymersolution was decanted and deionized water was added. The particles wereresuspended in the water for several minutes to rinse off any excesspolymer. The particles were then centrifuged once again and the waterremoved. Using this procedure, alternating layers of poly(diallyldimethylammonium chloride) and poly (styrene sulfonate) weredeposited onto the silica particles until 20 layers had been deposited.The outer layer comprised poly (styrene sulfonate), giving the particlesa negative surface charge.

Particles coated with polyelectrolyte multilayers were suspended inwater and pumped under pressure into fused silica capillaries with a solgel frit at the outlet. The particles accumulated into a packed bed atthe end of the capillary. Once the bed formed the particles adhered toeach other. The bed was stable, and no secondary retaining frit wasused.

The running electrolyte for electrophoresis experiments was phosphatebuffer at various concentrations and from about pH 4.0 to 8.0.Electrolyte solutions were made by adding 20 mM solutions of phosphoricacid to 20 mM phosphate salt solutions until the proper pH was achieved.The capillary length was 37 cm, length to detector 30 cm, and theapplied voltage was generally 15 kV. UV detection was performed at 254or 214 nm. Injection of the sample was performed electrokinetically atabout 5 kV for 5 sec, ca. 5 nL volume.

Acetone was used as a neutral electroosmotic flow marker, and2-phenoxypropionic acid (2-PPA) and lidocaine as negative and positivemarkers, respectively. Standard deviation values are reported as ±1σ.Electroosmotic mobility (μ_(eo)) is used here to quantify theelectroosmotic flow (EOF) and is given as the velocity of solvent flowper unit electric field strength (cm²V⁻¹s⁻¹).

Example 1 Advantage of Multilayer Over Single Layer

Separation of some representative basic proteins was performed using acapillary having a single layer of polyelectrolyte adsorbed and comparedto the separation obtained by using a capillary coated with a multilayercomprising six and a half layer pairs of alternating PDADMAC/PSS layers.Materials and methods were as described, except that for this exampleelectrophoresis was carried out at pH 4.0 in both types of layers. Thesingle layer PDADMAC was deposited from 10 mM polymer solution in3×10⁻³M NaOH. The multilayer was prepared as described above.

FIGS. 5 and 6 illustrate the single layer and multilayer separations,respectively. The first peak in each figure (unlabeled peak) represents2-phenoxypropionic acid, which was used as a negative marker. Thelabeled peaks are as follows: 1) α-chymotrypsinogen A; 2) ribonuclease;3) cytochrome C; and 4) lysozyme. Peaks labeled with prime numbersrepresent impurities or degradation products. Comparing the resultsdepicted in FIGS. 5 and 6, it can be noted that the elution order didnot change, and that peak shape is much more symmetrical and narrow forthe separation on the multilayer, indicating greater separationefficiency.

A single adsorbed layer of positive polyelectrolyte proved reasonablyeffective in promoting reversed flow CZE separations of basic proteins,as shown in FIG. 5. However, for separations at pH 6, the single layercolumn had to be “refreshed” by rinsing with polyelectrolyte in betweenruns. In addition, some tailing of the peaks was still observed evenafter rinsing.

Advantageously, capillaries coated with multilayers proved particularlyeffective in separating the proteins. FIG. 6 shows an electropherogramemploying silica coated with 6.5 layer pairs of PDADMAC/PSS. Elutionorder and migration times are comparable to the results of the singlelayer PDADMAC column shown in FIG. 5, however, the resolution hasimproved. In contrast, capillary zone electrophoresis failed to separatethe molecules when performed in bare, uncoated capillaries due toirreversible adsorption of the analyte proteins to the passage wall.

Example 2 Separation at Near Physiological pH

Separation was performed using a capillary having a single layer ofpolyelectrolyte adsorbed and compared to the separation obtained byusing a capillary coated with a multilayer comprising six and a halflayer pairs of alternating PDADMAC/PSS layers. Materials and methodswere as described, except that for this example electrophoresis wascarried out at pH 6.0 in both types of layers. The single layer PDADMACwas deposited from 10 mM polymer solution in 3×10⁻³ M NaOH. Themultilayer was prepared as previously described.

FIGS. 7 and 8 illustrate the single layer and multilayer separations,respectively. The first peak in each figure (unlabeled peak) represents2-phenoxypropionic acid, which was used as a negative marker. Thelabeled peaks are as follows: 1) α-chymotrypsinogen A; 2) ribonuclease;3) cytochrome C; and 4) lysozyme. Peaks labeled with prime numbersrepresent impurities or degradation products.

Comparing the results illustrated, it can be noted that the elutionorder again did not change. The multilayer coating behaved asefficiently at pH 6.0 as at pH 4.0. It is well known that at close toneutral pH separation efficiency is expected to decrease, probably dueto adsorption of analytes to uncovered silanol groups on the passagewalls. Conversely, proteins tend to degrade at lower pH values, with theresulting degradation products tending to clutter sensor readings, asnoted above. The results illustrated in FIG. 8 for separation on themultilayer at pH 6.0 show that peak height and peak area for impuritiesare reduced, indicating fewer degradation products present in the sampleanalyzed. Protein separation and reproducibility of migration times werecomparable to results obtained at pH 4.0. Analyte peak shape continuesfairly symmetrical and narrow for the separation on the multilayer,whereas the single layer separation indicates less resolution of peaks.

Example 3 Separation of Neutral Molecules

Neutral molecules, which generally tend to migrate together, were usedto test separation using a multilayer and compared to an uncoated, barecapillary tube. FIG. 9 illustrates separation results, the solid lineindicating separation on a multilayer prepared as described above, andthe dashed line indicating the result with the uncoated capillary.Separation conditions were as follows: 37 cm capillary length, with 30cm to the sensor, 50 μm internal diameter, 20 mM phosphate at pH 4.0 asrunning electrolyte, 5 kV operating voltage, electrokinetic injection ofthe sample, and detection by the sensor at 254 nm. FIG. 9 showsseparation of peaks for 1) acetone, and 2) phenol. The results indicatethat, while there was no separation in the bare silica capillary, therewas significant separation evident using the multilayer.

FIG. 10 illustrates separation peaks for 1) acetone, 2) fluorobenzene,3) phenol, and 4) p-cresol on a multilayer deposited as described, withthe modification of all layers being deposited in the presence of 0.5 MNaCl and 30% ethanol. Separation conditions were as follows: 37 cmcapillary length, with 30 cm to the sensor, 50 μm internal diameter, 20mM phosphate at pH 4.0 as running electrolyte, 15 kV operating voltage,electrokinetic injection of the sample, and detection by the sensor at254 nm.

The multilayer formed in the presence of ethanol did not appear to allowelution of a large neutral solute such as naphthalene. Presence ofethanol during multilayer deposition was shown to produce thickermultilayers, and it is theorized that a large neutral solute would tendto be retained within the multilayer. Therefore, a multilayer formed inthe presence of 0.5 M NaCl and 20% acetonitrile was tested forseparation and elution of naphthalene. Acetonitrile was used at a lowerconcentration and, since it is a slightly more polar molecule thanethanol, it was expected to form a thinner multilayer, therefore havinga smaller tendency to retain large neutral molecules. FIG. 11 showsseparation of peaks of 1) acetone, and 2) naphthalene. Separationconditions included: 80% 20 mM phosphate/20% acetonitrile as the runningelectrolyte and mobile phase; pH 6.0, 37 cm capillary length with 30 cmto the sensor, 50 μm internal diameter, 15 kV applied voltage,electrokinetic sample injection at 5 kV for 5 seconds, and detection at254 nm.

Example 4 Separation Using Multilayer Coated Particles

A capillary coated with a polyelectrolyte multilayer was prepared aspreviously described. Polyelectrolyte multilayer coated particles wereprepared also as described above, and packed into the capillary, asillustrated in FIG. 3.

Capillary electrochromatography was performed using water as the mobilephase. Acetone was used as the void time marker. Phenol and naphtholwere used as test compounds for the separation. Separation conditionswere as described in Example 3, above. Illustrative separation resultsfor phenol and acetone are shown in FIG. 12.

In the drawings and specification, there have been disclosed a preferredembodiment of the invention, and although specific terms are employed,the terms are used in a descriptive sense only and not for purposes oflimitation. The invention has been described in considerable detail withspecific reference to these illustrated embodiments. It will beapparent, however, that various modifications and changes can be madewithin the spirit and scope of the invention.

1. An apparatus for analytical separation of macromolecules, comprising:at least one capillary passage coated with a polyelectrolyte multilayerand having a first end electrically connected to said a positiveelectrode and a second end electrically connected to said a negativeelectrode to thereby generate an electrical field through said at leastone passage, wherein said polyelectrolyte multilayer comprises three ormore individual polyelectrolyte layers, each individual polyelectrolytelayer having a net electrical charge opposite from a net electricalcharge of an adjoining individual polyelectrolyte layer; and a sensorpositioned adjacent said at least one capillary passage for sensingmacromolecules.
 2. The apparatus of claim 1, wherein saidpolyelectrolyte multilayer comprises a plurality of organic compounds aspolyelectrolytes.
 3. The apparatus of claim 1, further comprising aplurality of particles positioned within said passage, wherein eachparticle is coated with a multilayer comprising a plurality ofpolyelectrolyte layers.
 4. The apparatus of claim 3, wherein saidparticles comprise fused silica.
 5. The apparatus of claim 1, whereinsaid passage comprises a substantially cylindrical void space having adiameter of from about five micrometers to about one hundredmicrometers.
 6. The apparatus of claim 1, wherein said passage isassociated with a body having the shape of a plate.
 7. The apparatus ofclaim 6, wherein said passage is positioned along a surface of saidbody.
 8. The apparatus of claim 6, wherein said passage is positionedwithin said body.